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Article: Unbiased Thiol-Labeling and Top-Down Proteomic Analyses Implicate Multiple Proteins in the Late Steps of Regulated Secretion.

Furber, Kendra L / Backlund, Peter S / Yergey, Alfred L / Coorssen, Jens R

2019 Volume 7, Issue 4

Abstract: Regulated exocytosis enables temporal and spatial control over the secretion of biologically active compounds; however, the mechanism by which ... ...

Abstract	Regulated exocytosis enables temporal and spatial control over the secretion of biologically active compounds; however, the mechanism by which Ca
Language	English
Publishing date	2019-09-27
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	2720995-7
ISSN	2227-7382
ISSN	2227-7382
DOI	10.3390/proteomes7040034
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Article ; Online: Pseudogenes regulate parental gene expression via ceRNA network.

An, Yang / Furber, Kendra L / Ji, Shaoping

Journal of cellular and molecular medicine

2017 Volume 21, Issue 1, Page(s) 185–192

Abstract: The concept of competitive endogenous RNA (ceRNA) was first proposed by Salmena and colleagues. Evidence suggests that pseudogene RNAs can act as a 'sponge' through competitive binding of common miRNA, releasing or attenuating repression through ... ...

Abstract	The concept of competitive endogenous RNA (ceRNA) was first proposed by Salmena and colleagues. Evidence suggests that pseudogene RNAs can act as a 'sponge' through competitive binding of common miRNA, releasing or attenuating repression through sequestering miRNAs away from parental mRNA. In theory, ceRNAs refer to all transcripts such as mRNA, tRNA, rRNA, long non-coding RNA, pseudogene RNA and circular RNA, because all of them may become the targets of miRNA depending on spatiotemporal situation. As binding of miRNA to the target RNA is not 100% complementary, it is possible that one miRNA can bind to multiple target RNAs and vice versa. All RNAs crosstalk through competitively binding to miRNAvia miRNA response elements (MREs) contained within the RNA sequences, thus forming a complex regulatory network. The ratio of a subset of miRNAs to the corresponding number of MREs determines repression strength on a given mRNA translation or stability. An increase in pseudogene RNA level can sequester miRNA and release repression on the parental gene, leading to an increase in parental gene expression. A massive number of transcripts constitute a complicated network that regulates each other through this proposed mechanism, though some regulatory significance may be mild or even undetectable. It is possible that the regulation of gene and pseudogene expression occurring in this manor involves all RNAs bearing common MREs. In this review, we will primarily discuss how pseudogene transcripts regulate expression of parental genes via ceRNA network and biological significance of regulation.
MeSH term(s)	Animals ; Gene Expression/genetics ; Gene Regulatory Networks/genetics ; Humans ; Pseudogenes/genetics ; RNA/genetics ; Response Elements/genetics
Chemical Substances	RNA (63231-63-0)
Language	English
Publishing date	2017-01
Publishing country	England
Document type	Journal Article ; Review
ZDB-ID	2074559-X
ISSN	1582-4934 ; 1582-4934 ; 1582-1838
ISSN (online)	1582-4934
ISSN	1582-4934 ; 1582-1838
DOI	10.1111/jcmm.12952
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Article ; Online: Biochemical Alterations in White Matter Tracts of the Aging Mouse Brain Revealed by FTIR Spectroscopy Imaging.

Furber, Kendra L / Lacombe, R J Scott / Caine, Sally / Thangaraj, Merlin P / Read, Stuart / Rosendahl, Scott M / Bazinet, Richard P / Popescu, Bogdan F / Nazarali, Adil J

Neurochemical research

2021 Volume 47, Issue 3, Page(s) 795–810

Abstract: White matter degeneration in the central nervous system (CNS) has been correlated with a decline in cognitive function during aging. Ultrastructural examination of the aging human brain shows a loss of myelin, yet little is known about molecular and ... ...

Abstract	White matter degeneration in the central nervous system (CNS) has been correlated with a decline in cognitive function during aging. Ultrastructural examination of the aging human brain shows a loss of myelin, yet little is known about molecular and biochemical changes that lead to myelin degeneration. In this study, we investigate myelination across the lifespan in C57BL/6 mice using electron microscopy and Fourier transform infrared (FTIR) spectroscopic imaging to better understand the relationship between structural and biochemical changes in CNS white matter tracts. A decrease in the number of myelinated axons was associated with altered lipid profiles in the corpus callosum of aged mice. FTIR spectroscopic imaging revealed alterations in functional groups associated with phospholipids, including the lipid acyl, lipid ester and phosphate vibrations. Biochemical changes in white matter were observed prior to structural changes and most predominant in the anterior regions of the corpus callosum. This was supported by biochemical analysis of fatty acid composition that demonstrated an overall trend towards increased monounsaturated fatty acids and decreased polyunsaturated fatty acids with age. To further explore the molecular mechanisms underlying these biochemical alterations, gene expression profiles of lipid metabolism and oxidative stress pathways were investigated. A decrease in the expression of several genes involved in glutathione metabolism suggests that oxidative damage to lipids may contribute to age-related white matter degeneration.
MeSH term(s)	Aging/physiology ; Animals ; Brain/metabolism ; Corpus Callosum/metabolism ; Mice ; Mice, Inbred C57BL ; Myelin Sheath ; Spectroscopy, Fourier Transform Infrared ; White Matter/metabolism
Language	English
Publishing date	2021-11-24
Publishing country	United States
Document type	Journal Article
ZDB-ID	199335-5
ISSN	1573-6903 ; 0364-3190
ISSN (online)	1573-6903
ISSN	0364-3190
DOI	10.1007/s11064-021-03491-y
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Article ; Online: Does Sirt2 Regulate Cholesterol Biosynthesis During Oligodendroglial Differentiation In Vitro and In Vivo?

Thangaraj, Merlin P / Furber, Kendra L / Sobchishin, LaRhonda / Ji, Shaoping / Doucette, J Ronald / Nazarali, Adil J

Cellular and molecular neurobiology

2018 Volume 38, Issue 1, Page(s) 329–340

Abstract: Sirtuin2 (SIRT2) is a deacetylase enzyme predominantly expressed in myelinating glia of the central nervous system (CNS). We have previously demonstrated that Sirt2 expression enhances oligodendrocyte (OL) differentiation and arborization in vitro, but ... ...

Abstract	Sirtuin2 (SIRT2) is a deacetylase enzyme predominantly expressed in myelinating glia of the central nervous system (CNS). We have previously demonstrated that Sirt2 expression enhances oligodendrocyte (OL) differentiation and arborization in vitro, but the molecular targets of SIRT2 in OLs remain speculative. SIRT2 has been implicated in cholesterol biosynthesis by promoting the nuclear translocation of sterol regulatory element binding protein (SREBP)-2. We investigated this further in CNS myelination by examining the role of Sirt2 in cholesterol biosynthesis in vivo and in vitro employing Sirt2
MeSH term(s)	Amino Acid Sequence ; Animals ; Cell Differentiation/physiology ; Cells, Cultured ; Cholesterol/biosynthesis ; Cholesterol/genetics ; Mice ; Mice, 129 Strain ; Mice, Inbred C57BL ; Mice, Knockout ; Neurogenesis/physiology ; Oligodendroglia/metabolism ; Sirtuin 2/physiology
Chemical Substances	Cholesterol (97C5T2UQ7J) ; Sirt2 protein, mouse (EC 3.5.1.-) ; Sirtuin 2 (EC 3.5.1.-)
Language	English
Publishing date	2018-01
Publishing country	United States
Document type	Journal Article
ZDB-ID	283404-2
ISSN	1573-6830 ; 0272-4340
ISSN (online)	1573-6830
ISSN	0272-4340
DOI	10.1007/s10571-017-0537-6
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Article ; Online: Organotypic Cultures from the Adult CNS: A Novel Model to Study Demyelination and Remyelination Ex Vivo.

Tan, Glaiza A / Furber, Kendra L / Thangaraj, Merlin P / Sobchishin, LaRhonda / Doucette, J Ronald / Nazarali, Adil J

Cellular and molecular neurobiology

2018 Volume 38, Issue 1, Page(s) 317–328

Abstract: Experimental models of multiple sclerosis (MS) have significantly advanced our understanding of pathophysiology and therapeutic interventions. Although in vivo rodent models are considered to most closely represent the complex cellular and molecular ... ...

Abstract	Experimental models of multiple sclerosis (MS) have significantly advanced our understanding of pathophysiology and therapeutic interventions. Although in vivo rodent models are considered to most closely represent the complex cellular and molecular disease states of the human central nervous system (CNS), these can be costly to maintain and require long timelines. Organotypic slice cultures maintain the cytotypic organization observed in the intact CNS, yet provide many of the experimental advantages of in vitro cell culture models. Cerebellar organotypic cultures have proven useful for studying myelination and remyelination, but this model has only been established using early postnatal tissue. This young brain tissue allows for neuro development ex vivo to mimic the 'mature' CNS; however, there are many differences between postnatal and adult organotypic cultures. This may be particularly relevant to MS, as a major barrier to myelin regeneration is age. This paper describes a modified protocol to study demyelination and remyelination in adult cerebellar tissue, which has been used to demonstrate neuroprotection with omega-3 fatty acids. Thus, adult cerebellar organotypic cultures provide a novel ex vivo platform for screening potential therapies in myelin degeneration and repair.
MeSH term(s)	Adult ; Age Factors ; Animals ; Central Nervous System/cytology ; Central Nervous System/metabolism ; Central Nervous System/pathology ; Cerebellum/cytology ; Cerebellum/metabolism ; Cerebellum/pathology ; Demyelinating Diseases/metabolism ; Demyelinating Diseases/pathology ; Humans ; Myelin Sheath/metabolism ; Organ Culture Techniques ; Remyelination/physiology
Language	English
Publishing date	2018-01
Publishing country	United States
Document type	Journal Article ; Review
ZDB-ID	283404-2
ISSN	1573-6830 ; 0272-4340
ISSN (online)	1573-6830
ISSN	0272-4340
DOI	10.1007/s10571-017-0529-6
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Article: Secretory vesicle cholesterol: Correlating lipid domain organization and Ca2+ triggered fusion.

Mahadeo, Mark / Furber, Kendra L / Lam, Simon / Coorssen, Jens R / Prenner, Elmar J

Biochimica et biophysica acta

2015 Volume 1848, Issue 5, Page(s) 1165–1174

Abstract: Membrane organization has received substantial research interest since the degree of ordering in membrane regions is relevant in many biological processes. Here we relate the impact of varying cholesterol concentrations on native secretory vesicle fusion ...

Abstract	Membrane organization has received substantial research interest since the degree of ordering in membrane regions is relevant in many biological processes. Here we relate the impact of varying cholesterol concentrations on native secretory vesicle fusion and the lateral domain organization of membrane extracts from these vesicles. Membranes of isolated cortical secretory vesicles were either depleted of cholesterol, had cholesterol loaded to excess of native levels, or were depleted of cholesterol but subsequently reloaded to restore native cholesterol levels. Lipid analyses confirmed cholesterol was the only species significantly altered by these treatments. Treated vesicles were characterized for their ability to undergo fusion. Cholesterol depletion resulted in a decrease of Ca2+ sensitivity and the extent of fusion, while cholesterol loading had no effect on fusion parameters. Membrane extracts were characterized in terms of lipid packing by surface pressure-area isotherms whereas the lateral membrane organization was analyzed by Brewster angle microscopy. While no differences in the isotherms were observed, imaging revealed drastic differences in domain size, shape and frequency between the various conditions. Cholesterol depletion induced larger but fewer domains, suggesting that domain coalescence into larger structures may disrupt the native temporal-spatial organization of the fusion machinery and thus inhibit vesicle docking, priming, and fusion. In contrast, adding excess cholesterol, or rescuing with exogenous cholesterol after sterol depletion, resulted in more but smaller domains. Therefore, cholesterol is an important membrane organizer in the process of Ca2+ triggered vesicular fusion, which can be related to specific physical effects on native membrane substructure.
MeSH term(s)	Animals ; Calcium Signaling ; Cholesterol/chemistry ; Cholesterol/deficiency ; Cholesterol/metabolism ; Intracellular Membranes/chemistry ; Intracellular Membranes/metabolism ; Intracellular Membranes/ultrastructure ; Membrane Fusion ; Microscopy ; Molecular Structure ; Pressure ; Secretory Vesicles/chemistry ; Secretory Vesicles/metabolism ; Secretory Vesicles/ultrastructure ; Stress, Mechanical ; Strongylocentrotus purpuratus
Chemical Substances	Cholesterol (97C5T2UQ7J)
Language	English
Publishing date	2015-05
Publishing country	Netherlands
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	60-7
ISSN	1879-2596 ; 1879-260X ; 1872-8006 ; 1879-2642 ; 1879-2618 ; 1879-2650 ; 0006-3002 ; 0005-2728 ; 0005-2736 ; 0304-4165 ; 0167-4838 ; 1388-1981 ; 0167-4889 ; 0167-4781 ; 0304-419X ; 1570-9639 ; 0925-4439 ; 1874-9399
ISSN (online)	1879-2596 ; 1879-260X ; 1872-8006 ; 1879-2642 ; 1879-2618 ; 1879-2650
ISSN	0006-3002 ; 0005-2728 ; 0005-2736 ; 0304-4165 ; 0167-4838 ; 1388-1981 ; 0167-4889 ; 0167-4781 ; 0304-419X ; 1570-9639 ; 0925-4439 ; 1874-9399
DOI	10.1016/j.bbamem.2015.02.005
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Article ; Online: Dissecting the mechanism of Ca2+-triggered membrane fusion: probing protein function using thiol reactivity.

Furber, Kendra L / Dean, Kwin T / Coorssen, Jens R

Clinical and experimental pharmacology & physiology

2010 Volume 37, Issue 2, Page(s) 208–217

Abstract: 1. Ca(2+)-triggered membrane fusion involves the coordinated actions of both lipids and proteins, but the specific mechanisms remain poorly understood. The urchin cortical vesicle model is a stage-specific native preparation fully enabling the directly ... ...

Abstract	1. Ca(2+)-triggered membrane fusion involves the coordinated actions of both lipids and proteins, but the specific mechanisms remain poorly understood. The urchin cortical vesicle model is a stage-specific native preparation fully enabling the directly coupled functional-molecular analyses necessary to identify critical components of fast triggered membrane fusion. 2. Recent work on lipidic components has established a direct role for cholesterol in the fusion mechanism via local contribution of negative curvature to readily enable the formation of transient lipidic fusion intermediates. In addition, cholesterol- and sphingomyelin-enriched domains regulate the efficiency of fusion by focally organizing other components to ensure an optimized response to the triggering Ca(2+) transient. 3. There is less known about the identity of proteins involved in the Ca(2+)-triggering steps of membrane fusion. Thiol reagents can be used as unbiased tools to probe protein functions. Comparisons of several thiol-reactive reagents have identified different effects on Ca(2+) sensitivity and the extent of fusion, suggesting that there are at least two distinct thiol sites that participate in the fusion mechanism: one that regulates the efficiency of Ca(2+) sensing/triggering and one that may function during the membrane merger event itself. 4. To identify the proteins that regulate Ca(2+) sensitivity, the fluorescent thiol reagent Lucifer yellow iodoacetamide was used to potentiate fusion and simultaneously tag the proteins involved. Ongoing work involves the isolation of cholesterol-enriched membrane fractions to reduce the complexity of the labelled proteome, narrowing the number of candidate proteins.
MeSH term(s)	Animals ; Calcium/metabolism ; Cholesterol/chemistry ; Cholesterol/metabolism ; Isoquinolines/chemistry ; Membrane Fusion ; Membrane Proteins/chemistry ; Membrane Proteins/metabolism ; Protein Interaction Domains and Motifs ; Sea Urchins ; Secretory Vesicles/chemistry ; Secretory Vesicles/physiology ; Sphingomyelins/chemistry ; Sphingomyelins/physiology ; Sulfhydryl Compounds/chemistry ; Sulfhydryl Compounds/metabolism
Chemical Substances	Isoquinolines ; Membrane Proteins ; Sphingomyelins ; Sulfhydryl Compounds ; lucifer yellow (9654F8OVKE) ; Cholesterol (97C5T2UQ7J) ; Calcium (SY7Q814VUP)
Language	English
Publishing date	2010-02
Publishing country	Australia
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	189277-0
ISSN	1440-1681 ; 0305-1870 ; 0143-9294
ISSN (online)	1440-1681
ISSN	0305-1870 ; 0143-9294
DOI	10.1111/j.1440-1681.2009.05278.x
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Article ; Online: Advances in the treatment of relapsing-remitting multiple sclerosis: the role of pegylated interferon β-1a.

Furber, Kendra L / Van Agten, Marina / Evans, Charity / Haddadi, Azita / Doucette, J Ronald / Nazarali, Adil J

Degenerative neurological and neuromuscular disease

2017 Volume 7, Page(s) 47–60

Abstract: Multiple sclerosis (MS) is a progressive, neurodegenerative disease with unpredictable phases of relapse and remission. The cause of MS is unknown, but the pathology is characterized by infiltration of auto-reactive immune cells into the central nervous ... ...

Abstract	Multiple sclerosis (MS) is a progressive, neurodegenerative disease with unpredictable phases of relapse and remission. The cause of MS is unknown, but the pathology is characterized by infiltration of auto-reactive immune cells into the central nervous system (CNS) resulting in widespread neuroinflammation and neurodegeneration. Immunomodulatory-based therapies emerged in the 1990s and have been a cornerstone of disease management ever since. Interferon β (IFNβ) was the first biologic approved after demonstrating decreased relapse rates, disease activity and progression of disability in clinical trials. However, frequent dosing schedules have limited patient acceptance for long-term therapy. Pegylation, the process by which molecules of polyethylene glycol are covalently linked to a compound, has been utilized to increase the half-life of IFNβ and decrease the frequency of administration required. To date, there has been one clinical trial evaluating the efficacy of pegylated IFN. The purpose of this article is to provide an overview of the role of IFN in the treatment of MS and evaluate the available evidence for pegylated IFN therapy in MS.
Language	English
Publishing date	2017-03-24
Publishing country	New Zealand
Document type	Journal Article ; Review
ISSN	1179-9900
ISSN (online)	1179-9900
DOI	10.2147/DNND.S71986
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Article ; Online: RNA-binding Protein Quaking Stabilizes

Thangaraj, Merlin P / Furber, Kendra L / Gan, Jotham K / Ji, Shaoping / Sobchishin, Larhonda / Doucette, J Ronald / Nazarali, Adil J

The Journal of biological chemistry

2017 Volume 292, Issue 13, Page(s) 5166–5182

Abstract: Myelination is controlled by timely expression of genes involved in the differentiation of oligodendrocyte precursor cells (OPCs) into myelinating oligodendrocytes (OLs). Sirtuin 2 (SIRT2), a ... ...

Abstract	Myelination is controlled by timely expression of genes involved in the differentiation of oligodendrocyte precursor cells (OPCs) into myelinating oligodendrocytes (OLs). Sirtuin 2 (SIRT2), a NAD
MeSH term(s)	Animals ; Cell Differentiation ; Gene Expression Regulation ; Mice ; Oligodendroglia/cytology ; Protein Binding ; Protein Isoforms/metabolism ; RNA Stability ; RNA, Messenger/metabolism ; RNA-Binding Proteins/metabolism ; RNA-Binding Proteins/physiology ; Response Elements ; Sirtuin 2/genetics
Chemical Substances	Protein Isoforms ; Qk protein, mouse ; RNA, Messenger ; RNA-Binding Proteins ; Sirt2 protein, mouse (EC 3.5.1.-) ; Sirtuin 2 (EC 3.5.1.-)
Language	English
Publishing date	2017-02-10
Publishing country	United States
Document type	Journal Article
ZDB-ID	2997-x
ISSN	1083-351X ; 0021-9258
ISSN (online)	1083-351X
ISSN	0021-9258
DOI	10.1074/jbc.M117.775544
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Article ; Online: Sirt2 epigenetically down-regulates PDGFRα expression and promotes CG4 cell differentiation.

Fang, Na / Cheng, Junjun / Zhang, Chu / Chen, Keyuan / Zhang, Chenyu / Hu, Zichao / Bi, Ran / Furber, Kendra L / Thangaraj, Merlin / Nazarali, Adil J / Ji, Shaoping

Cell cycle (Georgetown, Tex.)

2019 Volume 18, Issue 10, Page(s) 1095–1109

Abstract: We have previously found that Sirt2 enhanced the outgrowth of cellular processes and MBP expression in CG4 cells, where Sirt2 expression is suppressed by transcription factor Nkx2.2. However, the detailed mechanism of Sirt2 facilitating oligodendroglial ... ...

Abstract	We have previously found that Sirt2 enhanced the outgrowth of cellular processes and MBP expression in CG4 cells, where Sirt2 expression is suppressed by transcription factor Nkx2.2. However, the detailed mechanism of Sirt2 facilitating oligodendroglial cell differentiation remained unclear. In the present study, we observed that Sirt2 partially translocated into the nuclei when CG4 cells were induced to differentiate. Sirt2 was detected at the CpG island of PDGFRα promoter via ChIP assay during the cells differentiation process rather than during the state of growth. Sirt2 deacetylated protein(s) bound to the promoter of PDGFRα and simultaneously appeared to facilitate histone3 K27 tri-methylation, both of which are suppressive signatures on gene transcription activation. In bisulfate assay, we identified that Sirt2 significantly induced DNA methylation of PDGFRα promoter compared with the control. Consistently, Sirt2 overexpression down-regulated PDGFRα expression in CG4 cells. The knock-down of PDGFRα or Sirt2 over-expression repressed cell proliferation, but knock-down of Sirt2 promoted cell proliferation. Taken together, Sirt2 translocated into the nuclei while the cells initiated a differentiation process, facilitating CG4 cell differentiation partially through epigenetic modification and suppression of PDGFRα expression. The repression of PDGFRα expression mediated by Sirt2 appeared to facilitate a transition of cellular processes, i.e. from a proliferating progenitor state to a post-mitotic state in CG4 cells.
MeSH term(s)	Acetylation ; Active Transport, Cell Nucleus ; Animals ; Cell Differentiation ; Cell Line ; Cell Proliferation ; CpG Islands ; DNA Methylation ; Epigenesis, Genetic ; Gene Knockdown Techniques ; HEK293 Cells ; Humans ; Mice ; NIH 3T3 Cells ; Promoter Regions, Genetic ; Rats ; Receptor, Platelet-Derived Growth Factor alpha/genetics ; Receptor, Platelet-Derived Growth Factor alpha/metabolism ; Sirtuin 2/analysis ; Sirtuin 2/genetics ; Sirtuin 2/physiology
Chemical Substances	Sirt2 protein, rat ; Receptor, Platelet-Derived Growth Factor alpha (EC 2.7.10.1) ; Sirtuin 2 (EC 3.5.1.-)
Language	English
Publishing date	2019-05-10
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2146183-1
ISSN	1551-4005 ; 1538-4101 ; 1554-8627
ISSN (online)	1551-4005
ISSN	1538-4101 ; 1554-8627
DOI	10.1080/15384101.2019.1609818
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